This invention pertains to the control of growth and metabolism in fed-batch bioreactors.
The control of growth and metabolism is important because the maximum possible growth rate of an organism is often not the optimal growth rate in terms of process productivity. The maximum growth rate can easily be achieved and maintained by keeping all of the metabolites at high concentrations, but a process control method is needed when it is desired to maintain a growth rate that is lower than the maximum. In some cases, the maximum growth rate is optimal. But in these cases the metabolism may not be optimal because excess substrate may be metabolized to toxic or undesired byproducts. Thus, a method for controlling metabolism is needed. In short, the central goal in fermentation process engineering is to optimize a process by controlling growth and metabolism.
A primary object of the present invention and of fermentation process control in general is to maintain constant and optimal concentrations of the carbon and nitrogen sources in the growth medium so that the maximum possible specific product formation rate can be maintained for extended periods. In this way, the process yield and productivity can be improved or optimized for a process or a phase of the process and the batch-to-batch consistency can be improved. This general approach applies for any process, whether the product is a primary metabolite, a secondary metabolite, or the biomass itself.
The biological rationale behind this general approach is as follows:
1) The specific product formation rate depends on the relative activities of the organism's various metabolic pathways, which are regulated based on PA0 2) The organism's energetic state and the relative intracellular availabilities of carbon and nitrogen, which are affected by PA0 3) The organism's growth rate and metabolic state, which are affected by PA0 4) The organism's consumption rates of the carbon and nitrogen sources, which are affected by PA0 5) The concentrations of the carbon and nitrogen sources in the medium. PA0 1) Using the best growth medium (carbon source, nitrogen source, precursors, and nutrients such as vitamins and minerals), PA0 2) Using the optimal temperature, pH, redox potential, agitation rate, aeration rate, ionic strength, osmotic pressure, water activity, hydrostatic pressure, etc., PA0 3) Using the optimal dissolved oxygen or carbon dioxide concentration, PA0 4) Using inducers and repressors, PA0 5) Varying the above in a time-optimal fashion, PA0 6) Minimizing the accumulation of byproducts that negatively impact the growth or metabolism of the organism, PA0 7) Genetically altering the organism using recombinant DNA or hybridoma technology, and PA0 8) Using auxotrophic mutants or mutants with altered regulatory systems. PA0 1) In fed-batch processes, PA0 2) By maintaining constant metabolite concentrations, PA0 3) Using feedback to minimize or avoid overfeeding and underfeeding, PA0 4) In a practical manner, PA0 5) Using measurements that are fast, accurate, robust, and autoclavable, and PA0 6) Without using on-line or at-line measurements of metabolite concentrations.
Thus, a process can be optimized by maintaining the carbon and nitrogen sources at concentrations associated with the maximum specific product formation rate.
This general approach to fermentation process control was shown for a fed-batch antibody process by H. Kurokawa et al., "Growth Characteristics in Fed-Batch Culture of Hybridoma Cells with Control of Glucose and Glutamine Concentrations," Biotechnol. Bioeng., Vol. 44, pp. 95-103 (1994). Antibody production was shown to depend on the growth rate and the metabolic state, i.e., the relative consumption rates of the carbon source glucose and the nitrogen source glutamine. The antibody production was improved by using at-line liquid chromatography to measure the concentrations of glucose and glutamine and by using an adaptive feeding algorithm to control these concentrations. By maintaining the optimal glucose and glutamine concentrations, growth and metabolism were controlled at points associated with a high product formation rate. However, at-line liquid chromatography is not fast enough or robust enough for routine industrial use.
The need for a more practical control method, based on this general approach, for a fed-batch antibiotic process was shown by A. Lounes et al., "Effect of Nitrogen/Carbon Ratio on the Specific Production Rate of Spiramycin by Strepiornyces anibofaciens," Process Biochemistry, Vol. 3 1, pp. 13-20 (1996). Spiramycin production was shown to depend on the growth rate and the metabolic state, i.e., the relative consumption rates of the carbon source glycerol and the nitrogen source ammonia. But without a practical control method, the maximum specific product formation rate was only maintained during a short phase of the process.
Other general approaches to improving product formation rates include
The method of the present invention is not intended as a substitute for these environmental and genetic approaches, but as a complement.
Another general approach to fermentation process control is the use of continuous processes. Growth and metabolism are easily controllable in continuous bioreactors such as the chemostat, the pH-stat, and the RAR-stat. The latter was disclosed by H. Shimamatsu et al., "Process for Continuous Cultivation of Protein-Producing Microorganisms," U.S. Pat. No. 4,021,304 (May 3, 1977) and shown by P. Agrawal, "An Experimental Study of Acid Production Rate Controlled Operations of a Continuous Fermentor," Bioprocess Eng., Vol. 4, pp. 183-190 (1989), and is also referred to as an APR-stat (acid production rate). In continuous processes, the volume is constant because fresh medium is added at the same rate that broth (medium plus biomass) is withdrawn. Because of convection (flow through the system), steady state with respect to substrate, nutrient, biomass, and product concentrations, and thus growth and metabolism, is easily attainable. Growth and metabolism are controlled through the substrate and nutrient concentrations in the fresh medium and through the dilution rate for the chemostat, the buffering capacity of the fresh medium for the pH-stat, or the RAR set point for the RAR-stat (in all continuous processes, the growth rate equals the dilution rate at steady state). However, continuous fermentors are generally not used by industry because of various practical concerns such as the increased risk of contamination and the desire for batch downstream processing. Instead, fed-batch fermentors are preferred even though steady state is much harder to reach because of the lack of convection (in fed-batch reactors, the dilution rate is much less than the growth rate because concentrated feeds are used to minimize the increase in the volume).
Perhaps the most direct method to control growth and metabolism for fed-batch fermentors would involve on-line measurement and feedback control of substrate concentrations, i.e., a nutristat. But unfortunately, good (fast, accurate, robust, and autoclavable) substrate sensors are not yet available.
A promising but less direct method to control growth and metabolism is to use automated at-line measurements of the substrate concentrations. This was done, as discussed above, for glucose and glutamine using liquid chromatography and an adaptive feeding algorithm by H. Kurokawa et al., Biotechnol. Bioeng., Vol. 44, pp. 95-103 (1994). This was also done for glucose using a YSI Model 2000 analyzer and an algorithm for predicting the substrate consumption rate by B. F. Bishop et al., "Process Control System for Fed-Batch Fermentation Using a Computer to Predict Nutrient Consumption," U.S. Pat. No. 5,595,905 (Jan. 21, 1997). However, it is an object of the present invention to control fermentation growth and metabolism without measuring the concentrations of the growth-limiting metabolites.
Another method to control growth and metabolism is to feed the growth-limiting substrate according to an exponential schedule. However, this method is open-loop and does not have feedback, so overfeeding or underfeeding can occur at the beginning of the process if the biomass concentration is not estimated accurately, although eventually a constant growth rate may be reached.
Another method to control growth and metabolism is to feed the growth-limiting substrate in proportion to the measured total amount of biomass in the bioreactor, as shown by W. Hibino et al., "Three Automated Feeding Strategies of Natural Complex Nutrients Utilizing On-Line Turbidity Values in Fed-Batch Culture: A Case Study on the Cultivation of a Marine Microorganism," J. Ferm. Bioeng., Vol. 75, pp. 443-450 (1993). However, although this method is feedforward, it does not have feedback, so overfeeding can occur if the proportionality factor is not chosen properly.
Another method to control growth and metabolism is to feed the growth-limiting substrate such that the measured specific oxygen uptake rate (SOUR) is maintained at a set point corresponding to the desired growth rate. This method uses feedback and is effective when the SOUR is sufficiently sensitive, such as at low growth rates in which the growth- and nongrowth-associated components of oxygen uptake are comparable.
Another method to control growth and metabolism is to feed the growth-limiting substrate such that the measured respiratory quotient (RQ), defined as the carbon dioxide transfer rate (CTR) divided by the oxygen uptake rate (OUR), is maintained at a set point corresponding to the desired growth rate, as shown by S. Aiba et al., "Fed Batch Culture of Saccharomyces cerevisiae: A Perspective of Computer Control to Enhance the Productivity in Baker's Yeast Cultivation," Biotechnol. Bioeng., Vol. 18, pp. 1001-1016(1976) and S. Aiba et al., "Process for Growing Yeast in High Yield," Japanese Patent Laid-Open No. 52125686 (Oct. 21, 1977). This method uses feedback and is effective when the RQ is sufficiently sensitive, such as near the maximum growth rate in certain processes such as the aerobic yeast process, in which the undesired production of ethanol is indicated by an increase in the RQ.